Association between intracellular adenosine triphosphate content of CD4+ T lymphocytes and mortality in sepsis patients: A prospective observational study

Abstract Objective This study aimed to link intracellular adenosine triphosphate content in CD4+ T lymphocytes (CD4+ iATP) with sepsis patient mortality, seeking a new predictive biomarker for outcomes and enhanced management. Methods 61 sepsis patients admitted to the Intensive Care Unit between October 2021 and November 2022 were enrolled. iATP levels were gauged using whole blood CD4+ T cells stimulated with mitogen PHA‐L. Based on CD4+ iATP levels (<132.24 and ≥132.24 ng/mL), patients were categorized into two groups. The primary endpoint was all‐cause mortality. To identify factors associated with mortality, both univariate and multivariate Cox proportional hazard analyses were conducted. Results Of the patients, 40 had high CD4+ iATP levels (≥132.24 ng/mL) and 21 had low levels (<132.24 ng/mL). In a 28‐day follow‐up, 21 (34.4%) patients perished. Adjusting for confounders like SOFA score, APACHE II score, lactic acid, and albumin, those with low CD4+ iATP had three‐ to fivefold higher mortality risk compared to high CD4+ iATP patients (61.9% vs. 20.0%; hazard ratio [95% confidence interval], Model 1: 4.515 [1.276–15.974], p = .019, Model 2: 3.512 [1.197–10.306], p = .022). CD4+ iATP correlated positively with white blood cell and neutrophil counts but not with lymphocytes, CD3, and CD4 counts. Conclusions Low CD4+ iATP levels were associated with a higher risk of mortality in sepsis patients. Measurement of CD4+ iATP may serve as a useful tool for identifying patients at a higher risk of mortality and could potentially provide a basis for clinical treatment. Further research is warranted to fully elucidate the underlying mechanisms of this association.


| INTRODUCTION
Sepsis, characterized by a systemic inflammatory response syndrome resulting from infection, remains a significant health concern with ICU mortality rates ranging from 6% to 30%, escalating to over 40% in patients with septic shock. 1,2Currently, there is a lack of reliable prognostic indicators for critically ill patients with infections, leading to a dearth of targeted interventions and treatment strategies.Commonly employed prognostic assessment methods, such as the Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation Score II (APACHE II), suffer from subjectivity and fail to provide objective and intuitive data. 3,4Therefore, establishing an improved prognostic warning mechanism for critically ill patients with infections has emerged as a significant area of research.
6][7] Apart from the initial hyperinflammatory response, a considerable number of sepsis patients develop sepsis-associated immunosuppression, which has been implicated in disease progression and adverse outcomes, 6,8,9 this immunosuppression is characterized by dysfunctional T cell responses, including apoptosis, 10 exhaustion, 11 and impaired effector function, 12 further exacerbating the disease's severity.0][21] However, these biomarkers have certain limitations.Lymphocyte counts, while readily accessible, lack specificity due to various confounding factors.3][24] Immunomodulators may improve immune function and improve prognosis, but the curative effect is not clear due to lack of good monitoring indicators.Thus, further research is necessary to identify and validate biomarkers capable of accurately monitoring immune status, predicting sepsis outcomes, and feasible for clinical implementation.
The CD4+ cell ATP release assay evaluates the level of intracellular adenosine triphosphate (iATP) in purified CD4 + T lymphocytes after being stimulated with phytohemagglutinin-L (PHA-L).This technique has been developed to evaluate cell-mediated immune function, forecast the risk of infection, and monitor the rejection of solid organ transplants. 25,26Regarding severe infections, certain studies have indicated that within the initial 24 h of ICU admission, there is a notable difference in iATP levels within CD4 + T lymphocytes between survivors and non-survivors.The former group displays higher levels according to those studies. 26,27However, no further analysis was carried out on its correlation with the absolute counts of CD4 + T lymphocytes and its predictive value on the risk of all-cause mortality.The objective of this study is to investigate the prognostic significance of CD4 + T lymphocyte iATP levels in critically ill patients with infections.Additionally, it aims to examine the relationship between elevated ATP levels and the outcomes of infection.The findings of this research hold promise in contributing new insights toward establishing a prognostic warning mechanism for critically ill patients with infections in clinical practice.

| Study population
A total of 65 consecutive sepsis patients who met the criteria were admitted to the intensive care unit (ICU) of The Third Affiliated Hospital of Sun Yat-sen University between October 2021 and November 2022.Among them, four patients were excluded due to the lack of CD4 + iATP results.Finally, a total of 61 sepsis patients were included in this study.Based on their CD4 + iATP levels, they were divided into a high CD4 + iATP group and a low CD4 + iATP group (CD4 + iATP <132.24ng/mL and ≥132.24ng/mL).Throughout the follow-up period, there were no dropouts among the patients (Figure 1).Sepsis was defined according to the consensus established by the Third International Task Force for Sepsis and Septic Shock (Sepsis-3 definition). 28,29The diagnosis of sepsis was established through a comprehensive evaluation that considered all accessible information, such as imaging results, response to antibiotics, and surgical findings.Inclusion criteria:patient age range 18-70 years with sepsis at the time of admission; SOFA ≥ 2; patients with measurements of CD4 + iATP within 48 h of admission.Patients younger than 18 years; pregnant and lactating patients; patients with autoimmune diseases or malignant tumors under immunosuppressive therapy; patients without measurements of CD4 + iATP; patients having uncontrolled hemorrhage or other progressive diseases who were not expected to survive 24 h and patients who died within 24 h were excluded.This study followed the principles established in the Declaration of Helsinki and received approval from the Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University (No. [2022]02-216-01).Written informed consent was obtained from all participants, allowing the utilization of their data for research purposes.

| Data collection
Baseline data, including demographic characteristics, comorbidities, results of laboratory tests, treatment methods, and medications prescribed at discharge, were obtained from the electronic clinical management system.Comorbidities were ascertained based on preadmission diagnoses or diagnoses made during hospitalization.Laboratory examination records, including CD4 + iATP levels, were derived from the initial collection of whole blood samples obtained upon admission to the ICU.

| Measurement of CD4 + iATP levels
CD4 + T cell iATP levels were measured using the luciferin-luciferase reaction, in accordance with the manufacturer's instructions (Immune cell function assay kit, AIMdex R , Guangzhou Leide Biosciences Co., Ltd.).To elaborate, 100 μL of whole blood was diluted fourfold and then incubated with 25 μL of PHA-L (8.75 ng/mL) for a time period of 15-18 h inside a 5% carbon dioxide incubator set at 37 ± 0.5°C.Following this, CD4 + T cells were isolated and purified from the aforementioned solution utilizing magnetic beads that had been conjugated with monoclonal antibodies specific to CD4 + T cells.To extract the iATP, a lysis buffer was added to the CD4 + T cell sample.The resulting cell lysate was thoroughly mixed with a luciferin/luciferase mixture, and the bioluminescent product was measured after a 5-min reaction using a luminometer (JR-I, Weihai Wego Biotech Co., Ltd.).To establish an iATP standard curve, various concentrations (0, 50, 100, 200, 400, and 800 ng/mL) of iATP calibrators were employed.A comparison was made between the iATP levels of CD4 + T cells-depleted and non-depleted samples to ensure that the readings were exclusively attributable to iATP produced by the isolated cells.

| Endpoint and clinical definition
The primary endpoint was 28 days of all-cause mortality.SOFA scores were employed to evaluate organ dysfunction in ICU patients. 30The SOFA scores were designed specifically for this purpose.Additionally, the APACHE II score, comprising age, the Chronic Health Index, and the Acute Physiology Score (APS), was used.The APS was derived from 12 physiological parameters, including vital signs, arterial blood gas measurements, laboratory results, the Glasgow Coma Scale, among others.The calculation of the APACHE II score followed the methodology described by Knaus et al. 31

| Statistical analysis
Patients were categorized into two groups based on their CD4 + iATP levels: iATP <132.24ng/mL and iATP ≥132.24ng/mL, determined as the optimal cutoff value (Supporting Information S1: Figure 1).The optimal cutoff value was determined by assessing the ROC curve to achieve the best balance between sensitivity and specificity.Normally distributed continuous variables were described as means ± standard deviation (SD) and compared using Student's t test.Non-normally distributed continuous variables were reported as medians (interquartile ranges [IQRs]) and were compared using Mann-Whitney U test.Categorical variables were expressed as numbers (percentages) and the chi-squared test was used for categorical variables.Correlations between CD4 + iATP and other immune-related parameters were assessed using the Spearman correlation test.The proportional hazards assumption was examined using Schoenfeld residuals.Time-to-event data were visualized using Kaplan-Meier curves, and survival between groups was compared using log-rank tests.Univariate and multivariate analyses were conducted using Cox proportional hazard regression to identify significant predictors of mortality.Model 1 included CD4 + iATP, APACHE II score, SOFA score, lactic acid, albumin level, and presence of septic shock, while Model 2 included CD4 + iATP, age, gender, and SOFA score and the application of CRRT.A significance level of two-sided p ≤ .05 was considered statistically significant for all analyses.SPSS 20.0 software and R software (version 4.2.2) were used for statistical analysis.

| Baseline characteristics
The baseline characteristics stratified by CD4 + iATP categories (CD4 + iATP <132.24  p = .023)and higher shock rate (66.67% vs. 30.00%,p = .006).There were no significant differences observed between the two groups in terms of gender, APACHE II scores, diabetes mellitus, lactate level (Lac), albumin levels, lymphocyte counts, CD4 + T cell count, IL-6, and other laboratory examinations.Supporting Information S1: Table 1 provides baseline characteristics categorized by survival status.

| Associations between CD4 + iATP and mortality
We further investigated the impact of CD4 + iATP levels on patient outcomes, we conducted a follow-up study spanning 28 days.During a follow-up of 28 days, a total of 21 (34.4%)patients died.The Kaplan-Meier curves based on CD4 + iATP categories provide a visual representation of the survival trends, exposing a substantial disparity in the cumulative survival proportions among distinct groups (p < .001).This disparity underscores the influence of CD4 + iATP levels on the overall survival experience and highlights the potential clinical relevance of this biomarker in predicting patient outcomes (Figure 3).Univariate Cox analyses indicated a statistically significant association between mortality  2).

| DISCUSSION
The measurement of iATP has been shown to provide a systemic representation of CD4 + T cell function. 32Our study demonstrated that low levels of CD4 + iATP were associated with an increased risk of death.Through the incorporation of various models, including SOFA score, APACHE II score, lactic acid, albumin level, and shock rate, we consistently observed that patients with CD4 + iATP levels below 132.24 ng/mL had a three to five times higher risk of mortality compared to those with CD4 + iATP levels above this threshold (Table 2).The average CD4 + iATP level among non-survivors was significantly lower than the normal range. 27The lower CD4 + iATP levels in non-survivors compared to survivors were associated with the 28-day mortality rate in sepsis patients (Figure 3).Hence, monitoring CD4 + iATP levels during the early stages of sepsis may prove beneficial for risk assessment in sepsis patients.The underlying reason for the observed association between lower CD4 + iATP levels and a poorer prognosis in sepsis patients remains uncertain, necessitating further investigation.Nevertheless, there are several potential explanations that warrant exploration.First, this decline in ATP content could be attributed to mitochondrial dysfunction, which can impair ATP production. 33,346][37] It is worth noting that studies have reported a switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis as a hallmark of T cell activation, enabling them to meet the metabolic demands of proliferation. 38Additionally, it is plausible that this decrease may signify lymphocyte bioenergetic failure, representing an additional form of organ dysfunction within the context of sepsis.Further research is required to elucidate the precise mechanisms underlying this association and provide a comprehensive understanding of the relationship between CD4 + iATP content and prognosis in sepsis patients.
The lack of correlation between CD4 + iATP and CD4 + T cell or lymphocyte count observed in our study is an interesting finding (Figure 2), as it suggests that these two parameters may reflect different aspects of immune function.While CD4 + T cell count is a widely used measure of immune health, it may not fully capture the functional state of CD4 + T cells, as their activation and metabolic status can vary independently of their total count. 38,39The activation and metabolic status of CD4 + T cells can vary independently of their total count, and their capacity to synthesize and store ATP may also differ depending on their activation state and metabolic demands. 40Another intriguing observation is the close correlation between CD4 + iATP levels and mortality in sepsis, whereas isolated CD4 + T cell count or CD4 + iATP/CD4 + T cell count do not correlate with sepsis mortality.CD4 + iATP levels studied in our research actually result from a combination of cell quantity and individual CD4 + T cell ATP levels.We speculate that a single index may not fully represent a patient's CD4 cell immune function, as quantity and individual cell ATP levels can complement each other.Many patients exhibit insufficient CD4 + T cell count, but sufficient individual cell ATP levels, or vice versa, leading to maintenance of overall immune function within normal ranges.However, the specific mechanism behind this phenomenon requires further exploration.
Our study revealed a significant correlation between CD4 + iATP concentration and the counts of white blood cells and neutrophils in sepsis patients, consistent with the findings reported by Naderi et al. 26,41 However, a previous study indicated no association between iATP levels and white blood cell counts. 32Therefore, further research is needed to determine whether CD4+ iATP levels can effectively reflect systemic inflammation and innate immune responses.
It is important to note that iATP has been demonstrated to systematically represent the overall function of F I G U R E 3 Kaplan-Meier curves for all-cause mortality by CD4 + iATP categories.The comparison of survival rates according to the optimal cut-off iATP value in high ATP group and low ATP group.iATP, intracellular adenosine triphosphate.
T A B L E 2 Univariate and multivariate Cox regression models for CD4 + iATP levels and all-cause mortality.CD4 + T cells. 42The ATP measurement conducted using the PHA-L as a mitogen, which selectively activates CD4 + T cells. 32These findings suggest that iATP concentration values may serve as a reliable indicator of CD4 + T cell function and provide insights into the immune response in sepsis patients.The understanding of sepsis has evolved to recognize the involvement of both pro-inflammatory and antiinflammatory phases at different stages of the disease.Consequently, immunotherapy during sepsis has emerged as a significant area of research worldwide.4][45] In addition to these therapies, monitoring immune markers such as mHLA-DR and absolute lymphocyte counts has been investigated as a means of identifying septic patients with immunosuppression and guiding treatment decisions.However, there are still some studies that found that the predictive power of the HLA receptors has yielded mixed results.They were unable to find a good correlation between mHLA-DR downregulation or lymphocyte apoptosis and mortality when adjusted for confounders. 46,47Looking for reliable pro-inflammatory or antiinflammatory markers to evaluate the immune state is an urgent task.

Characteristic
Our study, along with other relevant reports, supports the notion that monitoring the concentrations of iATP produced by CD4 + T cells could be valuable in distinguishing between hypoimmune and hyperimmune states. 26This approach may aid in identifying patients at risk of infection or death, adjusting immunosuppressive agents according to CD4 + iATP content can help reduce mortality and infection in transplant patients, ultimately enhancing the effectiveness of immunotherapy interventions.By assessing CD4 + iATP levels, clinicians may be able to stratify patients and tailor immunomodulatory treatments accordingly, optimizing their response to therapy.This highlights the potential of iATP monitoring as a valuable tool in the management of sepsis-induced immunosuppression.
Our study has several limitations that should be acknowledged.First, the small sample size might introduce potential statistical biases or random errors.Additionally, our study was conducted at a single medical center and within a single ethnic group, which may limit the generalizability of the findings to other geographic locations, populations, or cultural settings.Despite adjusting for SOFA and other relevant indicators, there might be other unconsidered confounding factors that could influence the results.Furthermore, our study primarily focused on the relationship between CD4 + iATP and mortality and did not explore its association with other important clinical outcomes such as disease severity or length of hospital stay.This limitation restricts our comprehensive understanding and application of CD4 + iATP in the context of sepsis.
To gain a deeper understanding of the mechanisms underlying the association between CD4 + iATP content and sepsis prognosis, further research is warranted.This could involve designing multicenter randomized controlled trials to investigate the impact of immunomodulatory therapy on immune function and assessing the effects of changes in immune function on various clinical outcomes, including mortality, acute kidney injury, and duration of hospitalization.Such studies would contribute to advancing our knowledge and enhancing the clinical application of iATP monitoring in sepsis management.

| Conclusion
Our study demonstrates a strong association between low CD4 + iATP levels and increased mortality in sepsis patients.These findings highlight the potential of CD4 + iATP as a prognostic marker in sepsis management.To further validate its clinical utility, prospective clinical trials are warranted to evaluate the efficacy of CD4 + iATP-based immunologic interventions on patient outcomes.Such trials would provide valuable insights into the effectiveness of interventions targeting CD4 + iATP levels in improving outcomes for sepsis patients.constraints but can be obtained from the corresponding author upon making a reasonable request.

ETHICS STATEMENT
This study was approved by the ethical committee of the Third Affiliated Hospital of Sun Yat-sen University (No. [2022]02-216-01).All participants in this study provided written informed consent, allowing their clinical data to be utilized.The study adhered to the principles outlined in the Declaration of Helsinki.

F I G U R E 1
The study flow diagram.iATP, intracellular adenosine triphosphate.
T A B L E 1Note: Normally distributed parameters are reported as mean ± standard deviation, while non-normally distributed parameters are presented as median and interquartile range (IQR; Q1-Q3).Categorical variables are expressed as percentages.Chi-squared test was employed for variables expressed as percentages.Mann-Whitney U test was utilized for those represented as (IQR; Q1-Q3), and Student's t test was applied for variables represented as mean ± standard deviation.Abbreviations: APACHE II, Acute Physiologic Assessment and Chronic Health Evaluation-II; CRP, C-reactive protein; CRRT, continuous renal replacement therapy; iATP, intracellular adenosine triphosphate; IL-6, interleukin-6; Lac, lactate; LAC, blood lactate level; SOFA, Sequential Organ Failure Assessment score; WBC, white blood cell.and CD4 + iATP categories, SOFA score, APACHE II score, PaO 2 , lactic acid, and presence of septic shock.After adjusting for confounders (SOFA score, APACHE II score, lactic acid, albumin level, and shock rate), patients with low CD4 + iATP levels exhibited a 4.5-fold higher risk of mortality compared to those with high CD4 + iATP levels (Model 1: hazard ratio [HR]: 4.515, 95% confidence interval [CI]: 1.276-15.974,p = .019).Considering the clinical significance of variables, Model 2, which adjusted for age, gender, SOFA score, and the application of CRRT, revealed that low CD4 + iATP levels were indicative of a high risk of mortality, with an HR of 3.512 (95% CI: 1.197-10.306,p = .022)(Table Model I includes CD4 + iATP, APACHE II score, SOFA score, lactic acid, albumin level, and presence of septic shock; Model II includes CD4 + iATP, age, gender, SOFA score, and the application of CRRT.
Note:Abbreviations: APACHE II, Acute Physiologic Assessment and Chronic Health Evaluation-II; CRP, C-reactive protein; CRRT, continuous renal replacement therapy; iATP, intracellular adenosine triphosphate; Lac, blood lactate level; SOFA, Sequential Organ Failure Assessment score; WBC, white blood cell.